ABSTRACT
RESUMEN El filtrado de secuencias es un paso esencial sin importar el tipo de tecnología aplicada para la secuenciación de un genoma, en el cual las lecturas de baja calidad o una parte son eliminadas. En un ensamblado la construcción de un genoma se realiza a partir de la unión de lecturas cortas en cóntigos. Algunos ensambladores miden la relación que existe entre secuencias de una longitud fija (k-mer) que puede verse afectada por la presencia de secuencias de baja calidad. Un enfoque común para evaluar los ensamblados se basa en el análisis del número de cóntigos, la longitud del cóntigo más largo y el valor del N50, definido como la longitud del cóntigo que representa el 50 % de la longitud del conjunto. En este contexto, el presente estudio tuvo como objetivo evaluar el efecto del uso de lecturas crudas y filtradas en los valores de los parámetros de calidad obtenidos en el ensamblado del genoma de Bacillus altitudinis 19RS3 aislada de Ilex paraguariensis. Se realizó el análisis de calidad de ambos archivos de partida con el software FastqC y se filtraron las lecturas con el softwareTrimmomatic. Para el ensamblado se utilizó el software SPAdes y para su evaluación la herramienta QUAST. El mejor ensamblado para B. altitudinis 19RS3 se obtuvo a partir de las lecturas filtradas con el valor de k-mer 79, que generó 16 cóntigos mayores a 500 pb con un N50 de 931 914 pb y el cóntigo más largo de 966 271 pb.
ABSTRACT Sequence filtering is an essential step regardless of the type of technology applied for sequencing a genome, in which low-quality readings or a portion are eliminated. In an assembly, the construction of a genome is carried out from the union of short reads in contigs. Some assemblers measure the relationship between sequences of a fixed length (k-mer) that can be affected by the presence of low-quality sequences. A common approach to evaluating assemblies is based on the analysis of the number of contigs, the length of the longest contig, and the value of N50 defined as the length of the contig representing 50 % of the length of the assembly. In this context, the objective of this study was to evaluate the effect of the use of crude and filtered reads on the values of the quality parameters obtained from the genome assembly of Bacillus altituidinis 19RS3 isolated from Ilex paraguariensis. The quality analysis of both starting files was performed with the FastqC software and the readings were filtered with the Trimmomatic software. The SPAdes software was used for the assembly and the QUAST tool for its evaluation. The best assembly for B. altitudinis 19RS3 was obtained from the filtered readings with the value of k-mer 79, which generated 16 contigs greater than 500 bp with a N50 of 931 914 bp and the longest contig of 966 271 bp.
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Objective:The whole-genome sequencing and virulence characteristics analysis of a Klebsiella pneumoniae isolate that caused lower limb gas gangrene were performed to provide a reference for the comprehensive understanding of molecular virulence characteristics of K. pneumoniae causing severe community-acquired infection. Methods:The patient was admitted to the emergency department for treatment on March 13, 2018.The main clinical symptoms of the patients were high fever, gas gangrene of the left lower limb, and diabetic ketoacidosis. The pus specimen was collected for the bacterial culture, isolates identification and antimicrobial susceptibility testing. Hypermucoviscous phenotype was detected by string test. The whole genome of the isolate was sequenced and the multi-site sequence typing, capsular serotyping, plasmid characteristics, virulence and antimicrobial resistance genes of the isolate were analyzed. Plasmid curing and conjugation experiments were used to analyze plasmid characteristics. The virulence of the strain was assessed by serum killing and Galleria mellonella lethality assays. A two-sample t-test was used to compare the differences in the lethal dose of 50% (LD 50) between the tested strains and reference strains against the G. mellonella larvae. Results:K. pneumoniae strain KPN41053 was identified, it was only resistant to ampicillin and was negative for hypermucoviscous phenotype. Whole genome sequencing showed that the length of KPN41053 chromosome was 5 377 071 bp, belonging to ST660 type, and the capsular type was K16. A IncFIB(K)/HI1B virulence plasmid (207 506 bp) with a sequence that was highly similar to pLVPK was harbored by KPN41053. The plasmid carried a variety of virulence genes, among which rmpA and rmpA2 were pseudogenes. The plasmid could not be transferred horizontally by conjugation. The variation strain KPN41053_PC was obtained by plasmid curing. Serum killing analysis showed that KPN41053 was serum resistant (Grade 6), and KPN41053_PC was serum intermediately sensitive (Grade 3). The lgLD 50 of KPN41053 had no difference with that of the hypervirulent control strain (ST23-K1 type) ( t=0.32, P=0.765), and was significantly lower than that of KPN41053_PC ( t=5.97, P=0.004). Conclusions:KPN41053 was an atypical hypervirulent K. pneumoniae that belonged to ST660 but without a hypermucoviscous phenotype. The virulence plasmid harbored by KPN41053 was its key virulence factor. Hypervirulent K. pneumoniae can lead to community-acquired gas gangrene in diabetic patient, which deserves clinical attention.
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Sequencing technology has been greatly improved in terms of throughput and cost. The single-molecule nanopore DNA sequencing, one of the major branches of the third-generation sequencing technology, has made great contributions in the fields of medicine and life sciences due to its advantages of ultra-long reading length, real-time detection and direct detection of base methylation modification, etc. This article briefly describes the principle of nanopore sequencing technology, and discusses its application in clinical, animal, plant, bacterial and virus fields and its future development direction.
Subject(s)
Animals , Humans , Base Sequence , DNA , Chemistry , Genetics , Nanopore Sequencing , Nanopores , Research , Sequence Analysis, DNAABSTRACT
The ability to recognize and repair abnormal DNA structures is common to all forms of life. Physiological studies and genomic sequencing of a variety of bacterial species have identified an incredible diversity of DNA repair pathways. Despite the amount of available genes in public database, the usual method to place genomes in a taxonomic context is based mainly on the 16S rRNA or housekeeping genes. Thus, the relationships among genomes remain poorly understood. In this work, an approach of multiple gene sequence analysis based on genes of DNA repair pathway was used to compare bacterial genomes. Housekeeping and DNA repair genes were searched in 872 completely sequenced bacterial genomes. Seven DNA repair and housekeeping genes from distinct metabolic pathways were selected, aligned, edited and concatenated head-to-tail to form a super-gene. Results showed that the multiple gene sequence analysis using DNA repair genes had better resolution at class level than the housekeeping genes. As housekeeping genes, the DNA repair genes were advantageous to separate bacterial groups at low taxonomic levels and also sensitive to genes derived from horizontal transfer.
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0.05) or significantly enhanced(P0.05).In this case,the immunoadjuvant capability of CpG ODN was better than FQ68 genomes.Conclusion:CpG ODN or FQ68 genome enhances humoral and Th1 type of immune responses when packed into AIV oil emulsion vaccine,while the adjuvants do not show better adjuvanticity when separated from the oil emulsion vaccine.